The past decade has brought the approval of several new treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC) that extend overall survival. However, there is no cure for mCRPC, and development of novel therapeutic strategies is critical. Abiraterone and enzalutamide are two agents targeting the androgen receptor (AR) signaling pathway that extend patient survival, confirming the notion that AR remains a driver of mCRPC despite castrate levels of androgen ligands. Several mechanisms evolve during progression to mCRPC and resistance to abiraterone/enzalutamide that function to maintain AR signaling. These include amplification of AR, mutation of the ligand-binding domain of AR, and emergence of constitutively active alternatively spliced AR variants. This suggests that methods to ablate AR expression in mCRPC and deplete continued AR signaling may be effective in providing further survival benefit to patients. In this project, we will evaluate two approaches to ablate AR in mCRPC: AR antisense oligonucleotides (ASOs) and AR degraders. The AR ASO, IONIS-AR-2.5-Rx, has cleared a Phase I study and showed promising clinical responses in a heavily pretreated mCRPC population. Importantly, IONIS-AR-2.5-Rx targets full-length, mutant, and splice variant forms of AR. We have also undertaken a major effort to develop PROTAC (PROteolysis TArgeting Chimeric) AR degraders that function by targeting AR protein to an E3 ubiquitin ligase. Together, we hypothesize that ablation of AR, through ASOs or PROTAC degraders targeting AR, is a highly attractive therapeutic approach for mCRPC, and we will test this through the following Specific Aims: Aim 1: Evaluate IONIS-AR-2.5Rx, a next-generation AR ASO, in combination with enzalutamide in a Phase Ib/II clinical trial for the treatment of mCRPC. Here, we will continue the clinical development of IONIS-AR-2.5-Rx by performing a trial (ARRO-CITO) in combination with enzalutamide in chemotherapy-nave mCRPC patients. Molecular biomarkers of response will be identified through integrative clinical sequencing of tumors from the trial. This Aim will provide clinical proof-of-concept for AR ablative strategies in mCRPC. Aim 2: Develop potent, orally bioavailable AR degraders and study their mechanism of action. As a second approach to deplete AR levels, we will develop a PROTAC AR degrader through a stepwise drug development process and confirm its mechanism of action in vitro. Aim 3: Evaluate AR degraders in preclinical models of prostate cancer to select a candidate for a Phase I clinical trial in mCRPC. We will perform in vivo experiments in the first part of this Aim to assess pharmacokinetics, pharmacodynamics, and antitumor activity of our top AR degraders. A Phase I study will then be initiated with our lead compound in mCRPC patients, representing the first advancement of an AR degrader into clinical trials. These studies will lead to the development of two therapeutic approaches to ablate AR levels in mCRPC and prevent continued signaling through this driver pathway.